Fructooligosaccharides improves growth performance and intestinal epithelium function in weaned pigs exposure to Enterotoxigenic Escherichia coli

This study was conducted to explore the protective potential of Fructooligosaccharides (FOS) against Enterotoxigenic Escherichia coli (ETEC)-induced inammation and intestinal injury in weaned pigs. Methods Twenty-four weaned pigs were randomly assigned into three groups: (1) non-challenge (CON), (2) ETEC-challenge (ECON), and (3) ETEC challenge + 250 mg/kg FOS (EFOS). On day 19, non-challenged pigs were orally infused with sterilized culture while pigs in other groups were orally infused with ETEC (2.5 × 10 10 colony-forming units). After 3 days, pigs were slaughtered for sample collection. We show that ETEC-challenged signicantly reduced average daily gain (ADG); however, FOS improved the ADG (P < 0.05) and apparent digestibility of crude protein (CP), gross energy (GE), and ash in the ETEC-challenged pigs (P < 0.05). FOS reduced plasma concentrations of IL-1β and TNF-α, and elevated the concentrations of immunoglobulin A (IgA) and immunoglobulin M (IgM) (P < 0.05). Interestingly, FOS elevated villus height in duodenum, and elevated the ratio of villus height to crypt depth in the duodenum and ileum in the ETEC-challenged pigs (P < 0.05). Moreover, FOS increased lactase activity in the duodenum and ileum (P < 0.05). The activities of sucrase and alkaline phosphatase (AKP) were higher in EFOS group than in the ECON group (P < 0.05). Importantly, FOS up-regulated the expressions of critical genes in intestinal epithelium function such as zonula occludens-1 (ZO-1), L-type amino acid transporter-1 (LAT1), and cationic (CAT1) the glucose

Small intestine is the main site of nutrient digestion and absorption, and can serve as an important defense barrier against exogenous and endogenous harmful substances or pathogens [1]. Previous studies indicated that a wide variety of enteric pathogens such as pathogenic bacteria and viruses can induce acute diarrhea in mammalian animals, especially at their weaning period, resulting in mortality, dehydration, weight loss, and growth retardation [2][3][4]. In the last decades, antibiotics have been widely used for the preventing of diarrhea; however, long-term or overdose utilization of antibiotics increased the risk of developing drug resistance [5,6]. Therefore, alternatives for conventionally used antibiotic has attracted considerable research interest worldwide. The most widely studied alternatives include probiotics, prebiotics, enzymes, plant extracts, and nutraceuticals such as copper and zinc [7].
Oligosaccharides are carbohydrates of low degree of polymerization (DP) and low molecular weight composed of monosaccharides [8]. Oligosaccharides usually resist enzymatic hydrolysis and absorption in upper gastrointestinal tract, but can be fermented in large bowel by a number of bacteria [9]. Interestingly, non-digestible oligosaccharides have prebiotic activity, as they can promote body health by increasing populations of bene cial microbes and/or their metabolic activity [10]. FOS is composed of fructose and glucose units, which refers speci cally to short chains (3-6 units) of fructose units bound by β-(2-1) linkages that are attached to a terminal glucose unit [11]. Oligosaccharides including the FOS have been previously reported to improve growth performance and gut health in a variety of animal species [12]. For instance, FOS not only promoted the growth of weaning pigs, but also effectively reduced the colonization of pathogenic bacteria in the intestine [10].
ETEC is a major cause of diarrhea in neonatal and weaning pigs [13]. ETEC colonized in the intestine can release enterotoxins, which stimulates secretion of uid from the epithelial cells into the lumen, resulting in acute diarrhea [14]. On the other hand, weaning deprives neonatal pigs from passive immune protection of mother's milk, which increases their susceptibility to enterotoxigenic E. coli infection [15,16]. Although, numerous studies indicated that FOS has prebiotic effects that can inhibit the growth of pathogenic bacteria [17], only few reports indicated the in uences of FOS on the growth and intestinal health in weaned pigs exposure to ETEC challenge. In this study, we explored the effect of dietary FOS supplementation on growth performance, in ammatory response, intestinal epithelium function, and selected bacterial populations in weaned pigs upon ETEC challenge. This study will also provide convincing evidence on novel prebiotic effect of FOS and offer key insights into its potential mechanisms of action.

Animals, feeding and experimental design
All the procedures used in the animal experiment were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (no. 20181105). Twenty-four weaned pigs with an average initial body weight (6.30 ± 0.30 kg) pigs were randomly allotted into three treatment groups that included (1) non-challenged control (CON; pigs fed a basal diet and infused with sterilized Luria-Bertani culture);
The basal diet (Table 1) was formulated to meet the nutrient requirements recommended by the National Research Council 2012 [18]. Pigs were individually housed in metabolism cages (0.7 m × 1.5 m) with room temperature maintained at 25-28 °C and relative humidity controlled at 55-65%. All pigs were given an ad libitum access to fresh water and feeding. The trial lasted for 21 d. On day 19, pigs in the challenged groups were orally administered 150 mL of Luria-Bertani culture containing approximately 2.5 × 10 10 colony-forming units of ETEC (serotype O149:K91:K88ac; China Institute of Veterinary Drugs Control, Beijing, China) [19], while pigs in the non-challenged control group were administered an equal volume of sterilized Luria-Bertani culture. All pig's feeding consumption was measured daily and BW was measured on day 19 and 22 after 12-h fasting at the morning 8:00. The average daily body weight gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratio (G : F) were calculated. Total 100 excreta of each pig was weighted, and 10 mL of a 10% H 2 SO 4 solution was added to each 100 g of wet fecal sample, and subsequently stored in a sealed plastic bag at − 20 °C. At the end of the 4-d period, all fecal samples of each pig were thawed at room temperature and mixed thoroughly, and then dried at 65 °C for 48 h, after which they were ground to pass through a 1-mm screen and stored at − 20 °C for chemical analyses including dry matter (DM), crude protein (CP), ether extract (EE), ash, and gross energy. Blood samples were collected by venepuncture at 8:00 after 12 h of fasting. Then the samples were centrifuged at 3500 × g at 4 °C for 10 min [20]. After centrifugation, the plasma samples were collected and frozen at − 20 °C until analysis. After blood collection, pigs were euthanized with an intravenous injection of sodium pentobarbital and then slaughtered by exsanguination protocols, the mid points of the duodenum, jejunum and ileum of each pig were harvested and xed in 4% paraformaldehyde solution for morphological analyses. Besides, 2 g digesta from the cecum was immediately snap-frozen in liquid nitrogen, and stored at -80 ℃ for analysis of microbial DNA and Short-chain fatty acid. At the same time, mucosa samples were scraped with a scalpel blade from duodenum, jejunum and ileum segments and quickly-freezed using liquid N 2 , followed by the preservation at − 80 °C until further analysis for related enzyme activity and gene expression.

Measurement of growth performance
Piglets were weighed before the morning feed on day 1, 19, and 22 after 12-h fasting at the morning 8:00. Feeding intake was recorded, orts were collected and weighed daily. All feed was mash. Average daily feed intake(ADFI), average daily gain (ADG), and the ratio of gain to feed intake (G:F) were calculated.
Determination of the apparent total tract digestibility The apparent total tract digestibility (ATTD) was measured using acid-insoluble ash (AIA) as indicator [21]. The AIA in diets and faeces samples was determined by a method described by Chinese National Standard (GB/T23742). All samples were analyzed for DM, CP, EE, ash. The gross energy content of diets and fecal samples was determined using an adiabatic bomb calorimeter. Gross energy was determined using an automatic adiabatic bomb calorimeter (LECO,St. Joseph, Michigan, USA). The ATTD was calculated as (100-A1F2/A2F1 × 100), where A1 represents the AIA content of the diet; A2 represents the AIA content of faeces; F1 represents the nutrient content of the diet; F2 represents the nutrient content of faeces.
Intestinal morphology analysis About 1-cm segment of the small intestine (duodenum, jejunum and ileum) were mixed in 10% neutral buffered formaldehyde. The mixed tissue samples were dehydrated with normal saline and then embedded in para n. Cross sections of each sample were prepared, stained with haematoxylin and eosin (H&E) and then sealed by a neutral resin size. Ultrathin sections of the duodenal, jejunal and ileal samples were examined for the villus height and crypt depth with image processing and analysis system (Media Cybernetics, Bethesda, MD, USA). Villus height was calculated from the tip of the villi to the villuscrypt junction. Crypt depth was expressed as the invaginated depth between adjacent villi. A total of 10 intact, well-oriented crypt-villus units were analyzed in triplicate per segment. The ratio of villus height to crypt depth (V/C) was calculated from the values described above.

Determination of intestinal enzyme activities
After thawing, about 1 g of intestinal mucosa (including the duodenum, jejunum and ileum) was homogenized in ice-cold physiological saline at 1:9 and then centrifuged at 3000 × g, 4 °C for 15 min. The supernatant was assayed for protein content using the Bradford method, followed by measurement of the sucrase, lactase maltase and alkaline phosphatase (AKP) activities, in accordance with the instructions accompanying the respective kit (Nanjing Jiancheng Bioengineering Institute). The results were normalised to protein concentration and expressed as U/mg protein.

Intestinal microbial populations measurement
Intestinal microbial populations measurement Microbial DNA was extracted from the caecal digesta using the EZNA® Stool DNA kit (Omega Bio-Tek, Doraville, CA, USA), according to the manufacturer's instructions. Based on the 16S rRNA sequences of maximum species of each genus encountered in the pig intestinal tract downloaded from the National Center for Biotechnology Information (GenBank), European Molecular Biology Laboratory and DNA Data Bank of Japan, The uorescent quantitative speci c primers and probe for total E. coli, Lactobacillus, Bi dobacterium, and Bacillus (Table S1) were obtained from the Fierer et. al [22]. and Qi et. al [23] and and were commercially synthesised by Invitrogen (Shanghai, China). To quantify Bacillus, Bi dobacterium, E. coli and Lactobacillus, qPCR was carried out on a CFX96 Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA) using the RealMasterMix (Probe) kit (Tiangen Biotech Co., Ltd., Beijing, China). A volume of 25 µL, containing 12.5 µL of SuperReal PeMix (2.0×), 1 µL each of the forward and reverse primers, 1 µL of Fluorescent probe (20×), 1 µL of ROX Reference*3 Dye (50×), 2 µL of DNA and 6.5 µL of RNase-Free ddH 2 O were used in each reaction. The reactions were subjected to 1 cycle at 95 °C for 15 min, followed by 39 cycles at 95 °C for 3 s, 58 °C for 25 s and 72 °C for 60 s. The cycle threshold (Ct) values and baseline settings were determined by automatic analysis settings, and the copy numbers of the target group for each reaction were calculated from the standard curves, which were generated by constructing standard plasmids by a 10-fold serial dilution of plasmid DNA (1 × 10 1 to 1 × 10 9 copies/µL).

Volatile fatty acid (VFA) analysis
Samples of digesta from individual pigs were taken from the caecum.to measure the VFA concentration. The VFA concentrations in the digesta were determined using gas liquid chromatography according to the method described by Pierce et al [24]. Approximately 1.0 g of thawed cecal digesta was suspended in 1.5 mL of sterile milli-Q water in a centrifuge tube for 30 min. The entire sample was centrifuged at 12,000 × g for 10 min. 1 mL supernatant was transferred to a sterile tube, and then mixed with 0.2 mL 25% metaphosphoric acid and 23.3 µL 210 mmol/L crotonic acid simultaneously, the sterile tubes were centrifuged again for 10 min after placed the sterile tubes in ice-bath for 30 min. The mixture of 500 µL supernatant and 500 µL methanol was homogenized for 10 min in another sterile tube. After that, the mixture was centrifuged at 12,000 × g for 10 min at 4℃. The supernatant was injected into a gas  (Table S1).
Quantitative real-time polymerase chain reaction (PCR) was conducted to analyse the mRNA expression abundance of ZO-1, Occludin, FATP1, FATP4, CAT1, LAT1, SGLT1 and GLUT2 in the small intestinal mucosa using the CFX-96 real-time PCR detection system(Bio-Rad) and SYBR Premix Ex Taq II (Tli RNaseH Plus) reagents (TaKaRa, Dalian, China). The PCR reaction was run in a 10 µL reaction volume, which contained 5 µL of SYBR Premix Ex Taq II (Tli RNaseH Plus), 0.5 µL of each primer, 1 µL of the cDNA sample, and 3 µL of nuclease-free water. The PCR cycling parameters were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 57.5 °C for 30 s, and 72 °C for 5 min. A melting curve analysis was performed following each real-time quantitative PCR assay to con rm the Gene-speci c ampli cation products had been generated. The housekeeping gene β-actin was used as an internal control for normalization. The target gene mRNA expression level was calculated using the 2 −ΔΔCt method [25]. Each sample was simultaneously performed on the same PCR plate and three replicates were set up.

Statistical analysis
All data were subjected to one-way analysis of variance for a completely randomised design using the general linear model procedure of SPSS 24.0 (SPSS, Inc.), with each pig representing one experimental unit. Statistical differences among the treatments were separated by Duncan's multiple-range test. The results are expressed as with their standard errors. Statistical signi cance was set at P < 0.05 and 0.05 < P < 0.10 indicating a trend.

Results
Effect of FOS supplementation on growth performance and nutrient digestibility As shown in Table 2, there were no differences (P > 0.05) in ADG, average daily feed intake (ADFI), and feed e ciency (G:F) among the three treatments during days 1-18 (pre-challenge). ETEC challenge (during days 19-21) signi cantly reduced the ADG (P < 0.05) in the weaned pigs; however, the ADG was signi cant higher in the EFOS group than in the ECON group (P < 0.05). As compared to the CON and ECON group, dietary FOS supplementation signi cantly elevated the apparent digestibility of CP, GE, and Ash (P < 0.05). FOS supplementation did not signi cant in uence on the digestibility of DM (Table 3).   Table 4, ETEC challenge signi cantly increased the concentrations of TNF-α in the plasma (P < 0.05). However, dietary FOS supplementation signi cantly decreased the concentrations of plasma IL-1β and TNF-α in the pigs upon ECTC challenge (P < 0.05). Moreover, the concentrations of plasma IgA and IgG were lower in the ECON group than in the CON group (P < 0.05). However, the concentrations of plasma IgA and IgM were signi cantly higher in the EFOS group than in the ECON group (P < 0.05).  Figure. 1). As compared to the villus height was signi cant higher in the EFOS group than in the ECON group (P < 0.05). Interestingly, ETEC signi cantly decreased the ratio of villus height to crypt depth (V/C) in the duodenum and ileum (P < 0.05). As compared to the ECON group, FOS supplementation signi cantly increase the V/C ratio in the duodenum and ileum (P < 0.05).

Effect of FOS on enzyme activity of intestinal mucosa in weaned pigs upon ETEC challenge
As show in Table 6, ETEC challenge signi cantly decreased the mucosal activities of sucrase, lactase, and AKP in the duodenum (P < 0.05). The mucosal activity of lactase in the ileum was also lower in the ECON group than in the CON group (P < 0.05). However, FOS supplementation not only elevated duodenal the activities of sucrase, lactase, and AKP in the duodenum, but also elevated the ileal activity of lactase in the ETEC challenged pigs (P < 0.05). Values are means ± SEM, (n = 6), non-challenged pigs (CON, fed with basal diet), ETEC-challenged pigs (ECON, fed with basal diet), and FOS and ETEC-treated pigs (EFOS, fed with basal diet containing 250 mg/kg FOS challenged by ETEC). 2 a, b, c mean values within a row with unlike superscript letters were signi cantly different (P < 0.05). 3

AKP = alkaline phosphatase
Effect of FOS supplementation on microbial metabolites and intestinal populations in weaned pigs upon ETEC challenge As shown in Figure.2, ETEC challenge decreased the concentrations of acetic acid and propionic acid in cecal digesta (P < 0.05). However, FOS supplementation signi cantly elevated their concentrations in the ETEC challenged pigs (P < 0.05). The concentration of butyric acid was also higher in the EFOS group than in the ECON group (P < 0.05). ETEC challenge increased the abundance of E. coli in the cecal digesta; however, FOS supplementation signi cantly decreased the abundance of E. coli in the cecal digesta (Table 7). Moreover, FOS supplementation signi cantly elevated the abundance of bene cial micro ora such as the Bi dobacterium and Bacillus in the cecal digesta of ETEC-challenged pigs (P < 0.05). Effect of FOS supplementation on expressions of critical genes related to intestinal epithelium functions As shown in Figure.3, ETEC challenge decreased the expression levels of tight-junction protein ZO-1 in the duodenum. However, FOS supplementation not only elevated the expression levels of ZO-1 in the duodenum and jejunum, but also elevated the expression levels of occludin and FATP4 in the ileum (P < 0.05). Moreover, FOS supplementation signi cantly elevated the expression levels of LAT1 and CAT1 in the duodenum and ileum of the ETEC-challenged pigs (P < 0.05). The expression level of SGLT1 in the ileum, and the expression levels of GLUT2 in the jejunum and ileum were both higher in the EFOS group than in the ECON group (P < 0.05).

Discussion
Antibiotics are widely used for preventing post-weaning diarrhea in pig production industry [7]. However, long-term or overdose utilization of antibiotics may lead to developing of bacterial resistance and drug residues in the product [5,6]. Fructooligosaccharides are highly interesting prebiotic bres, resulting from the transfructosylating action of speci c microbial enzymes on sucrose, composed of one molecule of glucose linked to 2 to 4 fructose units at position β-1,2 that has been shown to improve weaned pigs' intestinal morphology and growth performance [10,12]. In the present study, we investigated the protective potential of FOS against ETEC-induced in ammation and intestinal injury in weaned pigs. We show that dietary FOS supplementation signi cantly improved the growth performance and apparent nutrient digestibility in pigs exposure to ETEC challenge. The result are consistent with previous studies on rats and pigs [26,27].
Infection with ETEC is a major cause of diarrheal disease in livestock production, especially in nursery pig cultivation [28,29]. TNF-α and IL-1β are two major pro-in ammatory cytokines involved in the in ammatory responses [30]. In the present study, ETEC challenge elevated the plasma concentration of TNF-α; however, FOS supplementation signi cantly decreased the concentrations of plasma IL-1β and TNF-α in the ETEC-challenged pigs, which suggested an ant-in ammatory role of FOS in regulating the ETEC-induced in ammation. The result is also consistent with previous study that mannan oligosaccharides added in the sow diet enhance their offspring intestinal immunity by decreasing in ammation [31]. Moreover, the plasma concentrations of IgA and IgG were elevated by FOS in the ETECchallenged pigs. Both the IgA and IgG are crucial index of body immune capacity and contribute to dealing with various pathogens. IgA is the main element of the humoral immune response that has been selected through evolution, together with innate mucosal defenses, to protest against microbial antigens at mucosal surfaces [32]. In general, IgG enhances mucosal homeostasis in addition to controlling noninvasive and invasive mucosal bacteria, and does so by reaching the lumen of mucosal organs upon binding to the epithelial transporter FcRn [33].
ETEC challenge signi cantly decreased the duodenal villus height, and reduced the ratio of V/C in the duodenum and ileum, indicating injury of the intestinal epithelium. The result is consistent with previous study that ETEC strains function through producing enterotoxins that act on the small intestines and lead to the secretion of uids and electrolytes, causing diarrhea and intestinal injury [34]. However, dietary FOS supplementation attenuated the intestinal injury by increasing the villus height and the ratio of V/C. The result is probably due to the prebiotic effects of oligosaccharides. For instance, the mannanoligosaccharides (MOS) were found to prevent the adhesion of enteric pathogens to intestinal epithelium, which signi cantly reduced their colonization and intestinal in ammation and injury [35]. Moreover, FOS was found to improve the intestinal morphology and signi cantly reduced the colonization of pathogenic bacteria in the intestine [36]. The bene cial effects of FOS supplementation on intestinal epithelium function has also been indicated by the mucosal enzyme activities, as FOS not only elevated duodenal the activities of sucrase, lactase, and AKP in the duodenum, but also elevated the ileal activity of lactase in the ETEC-challenged pigs. Sucrase and lactase are two important digestive enzymes involved in carbohydrate digestion, play crucial roles in the process of digestion in animals [37]. While, the AKP is an is another crucial endogenous enzyme expressed in the brush border, and is considered as an excellent marker enzyme involved in the primary digestive and absorptive processes of the small intestine [38].
In the present study, we also investigated the concentration of microbial metabolites and abundance of major bacterial populations in the cecum. As expected, FOS supplementation signi cantly elevated the concentrations of short chain fatty acids (SCFAs, e.g. acetic acid, propionic acid, and butyric acid) in the cecal digesta of ETEC-challenged pigs. The elevated SCFAs concentration is probably due to the fact that oligosaccharides cannot be digested in the upper digestive tract, but can be fermented by intestinal microorganisms in the lower diegstive tract [39]. Both the acetic and propionic acids can be used as energy substrates for peripheral tissues. While the butyrate not only act as a critical energy source for intestinal epithelial cells, but can also promote cell proliferation and differentiation [40]. Importantly, FOS decreased the abundance of E. coli, but signi cantly increased the abundance of Bi dobacterium and Bacillu in the cecum. Previous study has already indicated that oligosacchardes can not be utilized by pathogenic bacteria, but can be e ciently utilized by bene cial bacteria such as the Bi dobacterium and Bacillu [41]. The result is also consistent with previous studies on pigs and other animal species [42].
To gain insights into the mechanisms underlying the FOS-regulated intestinal health, we further investigated the expression levels of several critical genes involved in the intestinal epithelium functions.
Tight junctions play a critical role in maintaining the intestinal barrier integrity, which can prevent the paracellular diffusion of intestinal bacteria and other antigens across the epithelium [43]. Tight-junction proteins such as the ZO-1 and occludin are critical components for structural and functional organization of the tight junctions. In this study, FOS supplementation not only elevated the expression levels of ZO-1 in the duodenum and jejunum, but also elevated the expression levels of occludin and FATP4 in the ileum.
FATP4 is a small molecule protein that is responsible for transportation of fatty acids across the cell membranes [44]. Moreover, FOS supplementation signi cantly elevated the expression levels of nutrient transporters (LAT1, CAT1, SGLT1, and GLUT2) in the ETEC-challenged pigs. The LAT1 and CAT1 are tansporters that is responsible for transporting of the L-type and cationic amino acids, respectively [45]. While the SGLT1 and GLUT2 are closely associated with glucose absorption [46]. The elevated expressions of these nutrient transporters indicated an improved intestinal integrity and epithelium functions in pigs exposure to ETEC challenge.

Conclusions
The present study suggested that dietary FOS supplementation can attenuate the growth retardation in the weaned pigs exposure to ETEC challenge. Moreover, FOS can alleviate the ETEC-induced intestinal injury in the weaned pigs, which was associated with suppressing of the in ammatory responses and improving of the intestinal epithelium functions and intestinal microbiota. The bene cial effects of FOS on the growth performance and intestinal health should make it an attractive prebiotic that can be tentatively used for the substitution of antibiotics.

Declarations Data Availability and material
The datasets used to support the ndings of this study are available from the corresponding author upon request.

Con ict of Interest
The authors declare that there are no con icts of interest.